Nucleic Acid Biosynthesis Research Articles

9-(2'-Deoxy-2'-Fluoro-β-d-Arabinofuranosyl) Adenine Is a Potent Antitrypanosomal Adenosine Analogue That Circumvents Transport-Related Drug Resistance.

Current chemotherapy against African sleeping sickness, a disease caused by the protozoan parasite Trypanosoma brucei, is limited by toxicity, inefficacy, and drug resistance. Nucleoside analogues have been successfully used to cure T. brucei-infected mice, but they have the limitation of mainly being taken up by the P2 nucleoside transporter, which, when mutated, is a common cause of multidrug resistance in T. brucei We report here that adenine arabinoside (Ara-A) and the newly tested drug 9-(2'-deoxy-2'-fluoro-β-d-arabinofuranosyl) adenine (FANA-A) are instead taken up by the P1 nucleoside transporter, which is not associated with drug resistance. Like Ara-A, FANA-A was found to be resistant to cleavage by methylthioadenosine phosphorylase, an enzyme that protects T. brucei against the antitrypanosomal effects of deoxyadenosine. Another important factor behind the selectivity of nucleoside analogues is how well they are phosphorylated within the cell. We found that the T. brucei adenosine kinase had a higher catalytic efficiency with FANA-A than the mammalian enzyme, and T. brucei cells treated with FANA-A accumulated high levels of FANA-A triphosphate, which even surpassed the level of ATP and led to cell cycle arrest, inhibition of DNA synthesis, and the accumulation of DNA breaks. FANA-A inhibited nucleic acid biosynthesis and parasite proliferation with 50% effective concentrations (EC50s) in the low nanomolar range, whereas mammalian cell proliferation was inhibited in the micromolar range. Both Ara-A and FANA-A, in combination with deoxycoformycin, cured T. brucei-infected mice, but FANA-A did so at a dose 100 times lower than that of Ara-A.

Role of folate in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of chronic liver conditions that are characterized by steatosis, inflammation, fibrosis, and liver injury. The global prevalence of NAFLD is rapidly increasing in proportion to the rising incidence of obesity and type 2 diabetes. Because NAFLD is a multifaceted disorder with many underlying metabolic abnormalities, currently, there is no pharmacological agent that is therapeutically approved for the treatment of this disease. Folate is a water-soluble B vitamin that plays an essential role in one-carbon transfer reactions involved in nucleic acid biosynthesis, methylation reactions, and sulfur-containing amino acid metabolism. The liver is the primary organ responsible for storage and metabolism of folates. Low serum folate levels have been observed in patients with obesity and diabetes. It has been reported that a low level of endogenous folates in rodents perturbs folate-dependent one-carbon metabolism, and may be associated with development of metabolic diseases such as NAFLD. This review highlights the biological role of folate in the progression of NAFLD and its associated metabolic complications including obesity and type 2 diabetes. Understanding the role of folate in metabolic disease may position this vitamin as a potential therapeutic for NAFLD.

Role of CPS1 in Cell Growth, Metabolism and Prognosis in LKB1-Inactivated Lung Adenocarcinoma.

Liver kinase B1 ( LKB1 ) is a tumor suppressor in lung adenocarcinoma (LADC). We investigated the proteomic profiles of 45 LADC cell lines with and without LKB1 inactivation. Carbamoyl phosphate synthetase 1 (CPS1), the first rate-limiting mitochondrial enzyme in the urea cycle, was distinctively overexpressed in LKB1-inactivated LADC cell lines. We therefore assessed the role of CPS1 and its clinical relevance in LKB1-inactivated LADC. Mass spectrometric profiling of proteome and metabolome and function of CPS1 were analyzed in LADC cell lines. CPS1 and LKB1 expression in tumors from 305 LADC and 160 lung squamous cell carcinoma patients was evaluated by immunohistochemistry. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CPS1 and LKB1 expression. All statistical tests were two-sided. CPS1 knockdown reduced cell growth, decreased metabolite levels associated with nucleic acid biosynthesis pathway, and contributed an additive effect when combined with gemcitabine, pemetrexed, or CHK1 inhibitor AZD7762. Tissue microarray analysis revealed that CPS1 was expressed in 65.7% of LKB1-negative LADC, and only 5.0% of LKB1-positive LADC. CPS1 expression showed statistically significant association with poor overall survival in LADC (hazard ratio = 3.03, 95% confidence interval = 1.74 to 5.25, P < .001). Our findings suggest functional relevance of CPS1 in LKB1-inactivated LADC and association with worse outcome of LADC. CPS1 is a promising therapeutic target in combination with other chemotherapy agents, as well as a prognostic biomarker, enabling a personalized approach to treatment of LADC.

BIOLOGICAL ACTIVITY OF YERSINIA PSEUDOTUBERCULOSIS TOXINS

Study of effect of heat-labile (HLT) and thermostable (HST) lethal toxins of Yersinia pseudotuberculosis on the development of embryos of sea urchin Strongylocentrotus intermedius, processes of biosynthesis of nucleic acids and protein in embryo cells and activity of nucleoside- kinases of sea urchin. Materials-and methods. Y pseudotuberculosis strains 2517 (pYV-) and 512 (pYV48MD, pYV82MD) were used for isolation of HLT and HST Gametes and embryos of sea urchin S. intermediuswere used to carry out the experiments and isolate nucleoside-kinases. , Both of the studied toxins of Y pseudotuberculosis possessed, spermiotoxic effect and reduced fertilizing ability of sea urchin spermies. HLT LD₅₀ was 1 μg/ml, and HST - 2 μg/ml. Toxins affected the development of embryos of sea urchin resulting in severe morphologic damages, cessation ofthe development of embryos at early stages of embryogenesis, destruction of cells and death of embryos. Wherein; damaging effect of HLT was observed at lower concentrations compared with HST HLT inhibited DNA and RNA biosynthesis at concentrations of 1-2 μg/ml. HST did not affect biosynthesis of nucleic acids even at high concentrations, but inhibited protein biosynthesis in sea urchin embryos. HLT did not reduce the level of inclusion of labeled amino acids into embryo cells. HLT had inhibiting effect on the activity of thymidine- and uridine-kinase of sea urchin, whereas HST did not affect these enzymes. Both of Y pseudotuberculosis protein toxins affect the development of sea urchin embryos, however, mechanisms of action of HLT and HST on embryos and processes occurring in them differ.

Structure based virtual screening identifies Pranlukast as a potential inhibitor against Plasmodium falciparum Adenosine Deaminase enzyme

Malaria is a common infectious disease caused by Plasmodium species and the most lethal malarial parasite is P. falciparum. Considering the high malaria mortality rate and rapid emergence of multi drug resistant strains of malarial parasites necessitates the development of novel drugs. In this work, Adenosine Deaminase (ADA), the first enzyme of the purine salvage pathway was selected as a drug target due to its significant role in nucleic acid biosynthesis. Since the experimental structure of P. falciparum ADA is not available in Protein Data Bank (PDB), the target protein was modeled using comparative homology modeling approach. The overall quality of the model was good in terms of backbone dihedral angles distribution, ERRAT values, Profiles 3D score, knowledge based energy score and Z-score. The physico-chemical properties, secondary elements and active site pockets were analyzed using various in silico tools. Drug-like compounds retrieved from PubChem database was subjected to molecular docking with ADA. Pranlukast, the promising lead identified shows comparatively lower binding free energy compared to the control (5′-Methylthiocoformycin) and interacts with ADA through two hydrogen bonds as well as hydrophobic interactions. The molecular dynamics (MD) simulation studies revealed that Pranlukast binding does not have major alteration in the compactness and mobility of the protein backbone structure but it has an influence in the folding pattern and intramolecular hydrogen bonds formation. Thus, Pranlukast seems to be a potential inhibitor against P. falciparum ADA, which needs further validation by in vitro or in vivo experimental studies.

Nucleoside Derived Antibiotics to Fight Microbial Drug Resistance: New Utilities for an Established Class of Drugs?

Novel antibiotics are urgently needed to combat the rise of infections due to drug-resistant microorganisms. Numerous natural nucleosides and their synthetically modified analogues have been reported to have moderate to good antibiotic activity against different bacterial and fungal strains. Nucleoside-based compounds target several crucial processes of bacterial and fungal cells such as nucleoside metabolism and cell wall, nucleic acid, and protein biosynthesis. Nucleoside analogues have also been shown to target many other bacterial and fungal cellular processes although these are not well characterized and may therefore represent opportunities to discover new drugs with unique mechanisms of action. In this Perspective, we demonstrate that nucleoside analogues, cornerstones of anticancer and antiviral treatments, also have great potential to be repurposed as antibiotics so that an old drug can learn new tricks.

Comparative proteomic analyses for elucidating metabolic changes during EPS production under different fermentation temperatures by Lactobacillus plantarum Q823

Exopolysaccharide (EPS)-producing bacteria are of growing interest in industrial processes, mainly concerning food. Lactic acid bacteria are widely appreciated for their GRAS (generally recognized as safe) status and their ascertained or putative probiotic features. Detailed investigation on what happens at metabolic level during EPS production is scarce in the literature. The facultative heterofermenter Lactobacillus plantarum Q823 was studied in order to compare growth and EPS production at 30°C and 37°C. A higher growth rate was observed at 37°C, whereas, a significantly higher (tenfold increase) EPS amount was produced at 30°C. To understand the molecular mechanisms leading to the different EPS production in the two conditions, a comparative proteomic experiment was performed. The results of the in-gel proteomics revealed that: i) at 37°C a higher abundance of proteins involved in carbon catabolism and nucleic acid biosynthesis together with a significant amount of stress proteins was observed; ii) at 30°C the production of an atypical manganese-containing non-heme catalase (pseudocatalase) was increased, in agreement with previous data reporting that growth-rates of catalase negative Lactobacillus plantarum strains were greater than that of catalase positive strains. Taken together, all these findings provide further insights about the metabolic pathways stimulated during EPS production, and the mechanism that triggers EPS biosynthesis.

13 C-metabolic flux analysis of human adenovirus infection: Implications for viral vector production.

Adenoviruses are human pathogens increasingly used as gene therapy and vaccination vectors. However, their impact on cell metabolism is poorly characterized. We performed carbon labeling experiments with [1,2-13 C]glucose or [U-13 C]glutamine to evaluate metabolic alterations in the amniocyte-derived, E1-transformed 1G3 cell line during production of a human adenovirus type 5 vector (AdV5). Nonstationary 13 C-metabolic flux analysis revealed increased fluxes of glycolysis (17%) and markedly PPP (over fourfold) and cytosolic AcCoA formation (nearly twofold) following infection of growing cells. Interestingly, infection of growth-arrested cells increased overall carbon flow even more, including glutamine anaplerosis and TCA cycle activity (both over 1.5-fold), but was unable to stimulate the PPP and was associated with a steep drop in AdV5 replication (almost 80%). Our results underscore the importance of nucleic and fatty acid biosynthesis for adenovirus replication. Overall, we portray a metabolic blueprint of human adenovirus infection, highlighting similarities with other viruses and cancer, and suggest strategies to improve AdV5 production. Biotechnol. Bioeng. 2017;114: 195-207. © 2016 Wiley Periodicals, Inc.

Light Remodels Lipid Biosynthesis in Nannochloropsis gaditana by Modulating Carbon Partitioning between Organelles.

The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level.

RHIZOSPHERE MICROORGANISMS AND PHOSPHORUS AVAILABILITY FOR PLANTS

Phosphorus (P) is one of the major plant nutrients that directly or indirectly affect all biological processes. It is a key component of energy metabolism, and biosynthesis of nucleic acids and membranes. Phosphorus deficiency is considered to be one of the major limitations for crop production particularly in low-input agriculture systems around the world and nearly 80% of applied mineral P may be retained in the soil. Roots provide an interface for P moving from one living system—the soil—into another living system—the plant. Microbial processes in soil are important for the distribution of P between various inorganic and organic P fractions and subsequently for the potential availability of phosphate for plant absorption. These processes are predominantly manifest within the rhizosphere where readily-available C substrates are most abundant. So this review, focus on the contribution of soil microorganisms on the plant P nutrition through their capacity to increase the availability of soil P and its subsequent uptake by plants.

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-13C]glucose and [U-13C]glutamine, we apply for the first time 13C-Metabolic flux analysis (13C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and 13C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. 13C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning.

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.

Association of Genetic Polymorphisms in Genes Involved at the Branch Point of Nucleotide Biosynthesis and Remethylation with Down Syndrome Birth Risk: A Case-Control Study

DNA methylation and nucleic acid biosynthesis are two crucial phenomena for normal chromosomes segregation. From our earlier studies, MTHFR 677T individually and in combination with other gene polymorphisms, micronutrient deficiency and hyperhomocysteinemia was shown to be associated with risk in Down syndrome (DS) mothers. Remethylation and nucleic acid biosynthesis pathways are dependent on the activity of Methylenetetrahydrofolate reductase (MTHFR) and Thymidylate synthase (TYMS) respectively, competing for common substrate molecule 5,10-methelenetetrahydrofolate (5,10-MTHF). Role of MTHFD1 1958 G>A (affecting synthesis of 5,10-MTHF), MTHFR 677 C>T (affecting methylation), TYMS 5'UTR 28 bp repeat polymorphism and TYMS 3'UTR 6bp deletion polymorphism (affecting nucleic acid biosynthesis) in a cohort of 200 case mothers and 187 control mothers (also 146 case triads: mother, father, and child) were studied. We observed a significant association of MTHFR 677 C>T in a co-dominant model (p=0.0428) and dominant model (0.0194) as well as TYMS 5'UTR 28 bp repeat polymorphism in a recessive model (p=0.0005) and dominant model (0.0161). Genetic combination analysis revealed a significant additive effect of certain genotypic combinations (especially combination of MTHFR 677T and TYMS 2R alleles with other alleles or genotypes) in increasing risk. Weak linkage disequilibrium (LD) was observed between TYMS 5' and 3' UTR regions polymorphism in LD analysis. Transmission disequilibrium test (TDT) analysis revealed a consistent trend of preferential allele's transmission from parents. We concluded that genetic interaction of remethylation pathway and the nucleic acid metabolic pathway was significantly associated with risk factors for DS childbirth. However, replication studies are required to validate our observation in the population.

A pathway map of glutamate metabolism.

Glutamate metabolism plays a vital role in biosynthesis of nucleic acids and proteins. It is also associated with a number of different stress responses. Deficiency of enzymes involved in glutamate metabolism is associated with various disorders including gyrate atrophy, hyperammonemia, hemolytic anemia, γ-hydoxybutyric aciduria and 5-oxoprolinuria. Here, we present a pathway map of glutamate metabolism representing metabolic intermediates in the pathway, 107 regulator molecules, 9 interactors and 3 types of post-translational modifications. This pathway map provides detailed information about enzyme regulation, protein-enzyme interactions, post-translational modifications of enzymes and disorders due to enzyme deficiency. The information included in the map was based on published experimental evidence reported from mammalian systems.

Abstract LB-C20: Lead generation and optimisation of a series of novel glutaminase (GLS) inhibitors

Abstract Glutamine is an essential nutrient for cancer cells and is used to support cancer cell growth. Glutamine is converted to glutamate by the enzyme glutaminase, which is then converted into alpha-ketoglutarate, a metabolic intermediate within the TCA cycle which serves as a precursor for the biosynthesis of ATP, NADPH, fatty acids, glutathione, nucleic acids and amino acids. As such, we regarded glutaminolysis as a key pathway for therapeutic intervention. At the outset of the project there were several reported inhibitors of glutaminase known in the literature; however, they possess undesirable drug like properties. We describe here our efforts to discover novel inhibitors of glutaminase with a promising development profile. From a high throughput screen of approximately 900,000 compounds in the AstraZeneca collection, we were able to identify three series of glutaminase inhibitors. One of these series has been optimised to deliver cell potent, selective and orally bioavailable inhibitors of glutaminase 1. Citation Format: Mark D. Charles, Ceri Cairnduff, Joanna Brookfield, Verity Sabin, Tennyson Ekwuru, Neil Jones, Attilla Ting, James Smith, Willem Nissink, Ray Finlay, Steve Powell, Susan Critchlow, Richard Ward, Matthew Wood, Linette Ruston, Jon Winter, Ian Hollingsworth. Lead generation and optimisation of a series of novel glutaminase (GLS) inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C20.

Suitability of different growth substrates as source of nitrogen for sulfate reducing bacteria.

Sulfate reducing bacteria (SRB) mediated treatment of acid mine drainage is considered as a globally accepted technology. However, inadequate information on the role of nitrogen source in the augmentation of SRB significantly affects the overall treatment process. Sustenance of SRB depends on suitable nitrogen source which is considered as an important nutrient. This review focuses on the different nitrogen rich growth substrates for their effectiveness to support SRB growth and sulfate reduction in passive bioreactors. Compounds like NH4Cl, NH4HCO3, NO3 (-), aniline, tri-nitrotoluene, cornsteep liquor, peptone, urea, and chitin are reported to have served as nitrogen source for SRB. In association with fermentative bacteria, SRB can metabolize these complex compounds to NH4 (+), amines, and amino acids. After incorporation into cells, these compounds take part in the biosynthesis of nucleic acids, amino acids and enzyme co-factor. This work describes the status of current and the probable directions of the future research.

Gene expression profiling of rice seedlings in response to glutamine treatment

Glutamine, the most abundant free amino acid in humans (Curi et al., 2007 [1]), has many functions. In addition to protein, amino acid, and nucleic acid biosynthesis, glutamine also regulates the expression of genes related to metabolism, cell defense, and signal transduction in humans (Curi et al., 2007 [1]; Brasse-Lagnel et al., 2009 [2]). Glutamine is also one of the major forms of nitrogen in rice (Fukumorita and Chino, 1982 [3]). In addition to metabolic and nutritional effects, glutamine may function as a signaling molecule to regulate gene expression in plants. To this end, we used microarray analysis to identify genes that are rapidly induced by 2.5 mM glutamine in rice roots. The results revealed that glutamine induced the expression of at least 35 genes involved in metabolism, transport, signal transduction, and stress responses within 30 min (Kan et al., 2015 [4]). Here, we provide the details of the experimental procedure associated with our microarray data deposited in NCBI's Gene Expression Omnibus (GEO ID: GSE56770).

Profiling of the effects of antifungal agents on yeast cells based on morphometric analysis.

The incidence of fungal infection and evolution of multidrug resistance have increased the need for new antifungal agents. To gain further insight into the development of antifungal drugs, the phenotypic profiles of currently available antifungal agents of three classes-ergosterol, cell wall and nucleic acid biosynthesis inhibitors-were investigated using yeast morphology as a chemogenomic signature. The comparison of drug-induced morphological changes with the deletion of 4718 non-essential genes not only confirmed the mode of action of the drugs but also revealed an unexpected connection among ergosterol, vacuolar proton-transporting V-type ATPase and cell-wall-targeting drugs. To improve, simplify and accelerate drug development, we developed a systematic classifier that sorts a newly discovered compound into a class with a similar mode of action without any mutant information. Using well-characterized agents as target unknown compounds, this method successfully categorized these compounds into their respective classes. Based on our data, we suggest that morphological profiling can be used to develop novel antifungal drugs.

The expanding role of mTOR in cancer cell growth and proliferation.

The mechanistic/mammalian target of rapamycin (mTOR) is a conserved protein kinase that controls several anabolic processes required for cell growth and proliferation. As such, mTOR has been implicated in an increasing number of pathological conditions, including cancer, obesity, type 2 diabetes and neurodegeneration. As part of the mTOR complex 1 (mTORC1), mTOR regulates cell growth by promoting the biosynthesis of proteins, lipids and nucleic acids. Several mTORC1 substrates have been shown to regulate protein synthesis, including the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) and the ribosomal S6 kinases (S6Ks) 1 and 2. In this work, we focus on the signalling pathways that lie both upstream and downstream of mTORC1, as well as their relevance to human pathologies. We further discuss pharmacological approaches that target mTOR and their applications for the treatment of cancer.

Inhibition of nucleic acid biosynthesis makes little difference to formation of amphotericin B-tolerant persisters in Candida albicans biofilm.

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 μg ml(-1); biofilm cells, 1 μg ml(-1)) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 μg ml(-1) for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.